The long-term objectives of this research are to understand the mechanisms that regulate hair cell regeneration in the avian inner ear and to apply this knowledge to induce regeneration in the mammnalian cochlea. Ultimately, the goal of this research is to utilize hair cell regeneration to ameliorate deafness in humans. Studies from the previous two funding periods of this grant have shown that the auditory epithelium in the postembryonic avian cochlea is mitotically quiescent. However, in response to sound exposure or aminoglycoside administration, the nonsensory supporting cells emerge from quiescence and divide to produce new hair cells that contribute to the structural and functional recovery of the cochlea. In this proposal, we will examine how the proliferative state of supporting cells in the avian cochlea is regulated by activating signals from dying hair cells competing with inhibitory influences generated by connexin expression in gap junctions of differentiated supporting cells. Our hypothesis is that signals released during apoptosis in hair cells induce the supporting cells to exit quiescence and begin proliferating and that connexin43 expression suppresses proliferation in supporting cells of the normal cochlea. The specific aims of this proposal will examine: 1) the role of apoptosis in regulating hair cell death and supporting cell proliferation in the regenerating cochlea; and 2) the role of connexin gene expression in suppressing proliferation of supporting cells in the normal and 'regenerating cochlea. In the first aim we will identify key proteins in the apoptotic pathway, block apoptosis by surgically implanting miniosmotic pumps in birds that will deliver inhibitors of apoptosis directly to the inner ear, and determine how apoptosis regulates hair cell survival and supporting cell proliferation. In the second aim we will define the expression of connexin isoforms in the regenerating cochlea and we will disrupt the regulatory function of connexin43 in normal and regenerating cochlleae by surgically implanting miniosmotic pumps that will deliver inhibitors of gap junction connectivity to the inner ear. These specific aims will be addressed utilizing in situ hybridization and northern blot analysis for mRNA localization and quantification, immunoblots of cochlear protein expression, miniosmotic pump delivery of chemicals directly to the inner ear, and confocal microscopy for immunocytochemical localization of proteins in sectioned and wholemount cochleae.